Habituation in suspension-cultured soybean cells to thiamine and its precursors

When thiamine concentration in subculture medium was rapidlylowered to nil, soybean cells in suspension became necroticand stopped growing entirely. When it was gradually lowered,cell growth was vigorous until the concentration was reducedto 7.8?10^–3 mg/liter. The cells at this level of thiamineceased growing for a time, but prolonged culture in the samemedium resulted in the appearance of fresh white cells whichcould be easily distinguished from the old brown, necrotic cellsin the aggregates. These new cell lines could be subculturedwith further reduction in the thiamine supply, growing as largeraggregates of about 4 mm in diameter. New cell lines were similarly obtained by prolonged culturesin media containing a thiamine precursor; three lines appearedto be habituated to the pyrimidine moiety and one to the thiazolemoiety. The latter cell line could be subcultured without thiamineand its precursors for at least eight passages. These habituatedcells were characterized by the increase of the dry to freshweight ratio and by their growth in large aggregates. ¹Present address: Section of Phytochemical Research, Eisai Co.,Ltd., Kawashima, Gifu 483, Japan. (Received December 15, 1978; )     

Thiamine requirement of two different cultured cell lines of soybean

When supplied with 6-benzyladenine (0.5–5 mg/liter) insteadof thiamine, thiamine-requiring soybean cells (strain TU) couldgrow successively. The effect of cytokinin was much more remarkableat a relatively higher concentration of 2,4-dichlorophenoxyaceticacid (4 mg/liter) than at a lower concentration (0.5 mg/liter).Among calli initiated from soybean hypocotyls on a medium withoutthiamine, the thiamine-nonrequiring variant (strain G) was obtainedincidentally. As this cell line became green in light, it couldbe visually separated from the other necrotic tissues. StrainG cells could grow successively not only without thiamine butalso without phytohormones, auxin and cytokinin. This cell linehad relatively higher amounts of chlorophyll and thiamine, andgrew in rigid, large cell aggregates which differed from cellaggregates of the strain TU cell line. The thiamine requirementof plant cultured cells seems to be associated with the degreeof dedifferentiation of the cells rather than the kind of plant.In general, the higher the degree of redifferentiation of thecells, the higher is their thiamine level and the less theyrequire externally supplied thiamine. ¹Present address: Section of Phytochemical Research, Eisai Co.,Ltd., Kawashima, Gifu 483, Japan. (Received December 15, 1978; )     

Thiamine requirements of various plant cells in suspension culture

The thiamine requirements of cells of several plant speciesin suspension culture were investigated. Omission of thiaminefrom the medium leads to a rapid and complete cessation of growthin subcultures of soybean, tobacco and rice cells. The criticallevels of thiamine content in these cells are considered tobe in the vicinity of 0.6, 0.5 and 0.2 µg per g dry weight,respectively. Soybean cells can grow satisfactorily when suppliedwith an equimolar mixture of two thiamine precursors, pyrimidineand thiazole moieties. Neither moiety alone can substitute forthiamine. On the other hand, Rula and peanut cells can be successfullysubcultured for ten passages in the absence of externally suppliedthiamine. When grown without thiamine, Rula cells had an averagethiamine content of 0.5-0.6 µg in darkness and 3.5 µgper g dry weight in the light. The relationship between thethiamine requirement and the degree of dedifferentiation incultured cells is discussed. ¹ Present address: Section of Phytochemical Research, Researchand Development Division, Eisai Co., Ltd., Kawashima, Gifu 112,Japan. (Received December 24, 1975; )     

Germination of Sporangia of Phytophthora palmivora (Butl.) Butl.

Sporangia of Phytophthora palmivora germinated by either forminggerm tubes or producing zoospores. Two distinct modes of germ-tubedevelopment have been described. Sporangia in distilled waterformed zoospores at 10–34°C with an optimum at 22°Cbut germinated by means of germ tubes at 30 and 34°C only.Zoospore formation was inhibited to varying degrees by cocoapod extract, I.0 per cent (w/v) peptone and yeast extract, 100–500µg1^-1 thiamine, and by very low concentrations of severalamino acids, carbohydrates, and inorganic salts. Germ-tube formation was encouraged at 22°C by 1'0 per cent(w/v) peptone and yeast extract, by cocoa pod extract and exudate,10mM CaCl2, 1–10 mM MgSO₄. 7H₂O, 0.5 per cent (w/v) fructose,galactose, glucose, lactose, maltose, and sucrose, by 100 ppmarginine, aspartic acid, glutarnic acid, glycine, leucine, andtryptophane, and by 100–500 µg 1^-1 thiamine.     

Phosphoenolpyruvate Carboxylase of Maize Leaves: an Improved Method for Purification and Reduction of the Inhibitory Effect of Malate by Ethylene Glycol and Bicarbonate

Light-adapted and dark-adapted forms of phosphoenolpyruvatecarboxylase were purified from maize leaves by an improved methodthat included chromatography on Butyl-Toyopearl in the presenceof ethylene glycol. The inhibition by malate was relieved notonly by increasing concentrations of ethylene glycol but alsoby bicarbonate at pH 7.0. ¹Present address: NEOS Central Research Laboratory, 1-1 Ohike-machi,Kosei-cho, Shiga, 520-32 Japan. ²Present address: Asahi Medical Co., Ltd., 4-3400-I Asahimachi,Nobeoka, 882 Japan. ³Present address: Chugai Pharmaceutical Co., Ltd., 1-135 Komakado,Gotemba, 412 Japan.     

The Deduced Amino Acid Sequence of Phytochrome from Adiantum Includes Consensus Motifs Present in Phytochrome B from Seed Plants

A cDNA for the phytochrome of the fern Adiantum capillus-venerisL. was cloned and sequenced. The deduced phytochrome is 50{smalltilde}55% identical to phytochromes of seed plants, and 68%identical to Selaginella phytochrome. Regions resemble thosein previously characterized phytochromes from ferns, lower plantsand seed plants. ³Present address: Yamanouchi Pharmaceutical Co., Ltd., 21 Miyukigaoka,Tsukuba-shi, Ibaraki, 305 Japan ⁴Present address: Plant Growth Regulation Laboratory, The Instituteof Physical and Chemical Research (RIKEN), Hirosawa 2-1, Wako-shi,Saitama, 351-01 Japan ⁵Present address: Advanced Research Laboratory, Hitachi, Ltd.,Hatoyama, Saitama, 350-03 Japan     

Transport of thiamine in human intestine: mechanism and regulation in intestinal epithelial cell model Caco-2

The present studyexamined the intestinal uptake of thiamine (vitaminB₁) using the human-derivedintestinal epithelial cells Caco-2 as an in vitro model system.Thiamine uptake was found to be 1)temperature and energy dependent and occurred with minimal metabolicalteration; 2) pH sensitive;3)Na⁺ independent;4) saturable as a function ofconcentration with an apparent Michaelis-Menten constant of 3.18 ± 0.56 µM and maximal velocity of 13.37 ± 0.94 pmol · mgprotein¹ · 3 min¹;5) inhibited by the thiaminestructural analogs amprolium and oxythiamine, but not by unrelatedorganic cations tetraethylammonium, N-methylnicotinamide, and choline; and6) inhibited in a competitive mannerby amiloride with an inhibition constant of 0.2 mM. The role ofspecific protein kinase-mediated pathways in the regulation of thiamineuptake by Caco-2 cells was also examined using specific modulators ofthese pathways. The results showed possible involvement of aCa²⁺/calmodulin (CaM)-mediatedpathway in the regulation of thiamine uptake. No role for proteinkinase C- and protein tyrosine kinase-mediated pathways in theregulation of thiamine uptake was evident. These results demonstratethe involvement of a carrier-mediated system for thiamine uptake byCaco-2 intestinal epithelial cells. This system isNa⁺ independent and is differentfrom the transport systems of organic cations. Furthermore, aCaM-mediated pathway appears to play a role in regulating thiamineuptake in these cells.

     

Mechanism of thiamine uptake by human jejunal brush-border membrane vesicles

Thiamine, a water-soluble vitamin, is essential fornormal cellular functions, growth and development. Thiamine deficiency leads to significant clinical problems and occurs under a variety ofconditions. To date, however, little is known about the mechanism ofthiamine absorption in the native human small intestine. The objectiveof this study was, therefore, to characterize the mechanism of thiaminetransport across the brush-border membrane (BBM) of human smallintestine. With the use of purified BBM vesicles (BBMV) isolated fromthe jejunum of organ donors, thiamine uptake was found to be1) independent of Na⁺ but markedly stimulated byan outwardly directed H⁺ gradient (pH 5.5_in/pH7.5_out); 2) competitively inhibited by thecation transport inhibitor amiloride (inhibitor constant of 0.12 mM);3) sensitive to temperature and osmolarity of the incubation medium; 4) significantly inhibited by thiamine structuralanalogs (amprolium, oxythiamine, and pyrithiamine), but not byunrelated organic cations (tetraethylammonium,N-methylnicotinamide, or choline); 5) notaffected by the addition of ATP to the inside and outside of the BBMV;6) potential insensitive; and 7) saturable as afunction of thiamine concentration with an apparent Michaelis-Menten constant of 0.61 ± 0.08 µM and a maximal velocity of 1.00 ± 0.47 pmol · mg protein¹ · 10 s¹. Carrier-mediated thiamine uptake was also found inBBMV of human ileum. These data demonstrate the existence of aNa⁺-independent, pH-dependent, amiloride-sensitive,electroneutral carrier-mediated mechanism for thiamine absorption innative human small intestinal BBMV.

     

Biosynthetic mechanism of glycolate in Chromatium II. Enzymic mechanism of glycolate formation by a transketolase system

The mechanism for the biosynthesis of glycolate in the photosyntheticbacterium Chromatium was studied. The enzymic nature of glycolatesynthesis from fructose-6-P and ribose-5-P by a cell-free bacterialextract, strictly dependent on the presence of ferricyanidein the assay mixture, was established. Removal of thiamine pyrophosphatefrom the assay mixture did not alter the magnitude of glycolateformation. By examining the reaction products from ¹⁴C-fructose-6-Pand ¹⁴C-ribose-5-P, the operating mechanism involved in glycolateformation was judged to be the oxidative breakdown of the glycolaldehyde-transketolaseaddition product by ferricyanide. The role of transketolasein the oxidative formation of glycolate in the photosynthesizingcells of Chromatium is discussed. ¹ This is paper XXXI in the series "Structure and Function ofChloroplast Proteins". Paper XXX is reference (5). The researchwas supported in part by grants from the Ministry of Educationof Japan (986009), the Toray Science Foundation (Tokyo), andthe Naito Science Foundation(Tokyo). (Received May 22, 1975; )     

Fungal extracts that induce phytoalexins in sweet potato roots

Soluble extracts from mycelia and conidia of two strains ofCeratocyslis fimbriata induced formation of terpenes in sweetpotato root tissue. Factors inducing terpene formation are water-or 0.02 M KCl-soluble, heat stable, organic solvent-insoluble,and dialyzable, and have neither cationic nor anionic properties.They caused cellular injury of root tissue, accompanied by productionof ethylene. ¹This paper constitutes Part 115 of the Phytopathological Chemistryof Sweet Potato with Black Rot and Injury, and Contributionof Research Branch, Agriculture Canada, Winnipeg, Canada. Thiswork was supported in part by a grant from the Ministry of Education,Japan. ²Present address: Research Branch, Research Station, AgricultureCanada, Winnipeg, Manitoba, Canada. (Received July 27, 1974; )     

Oriented structure of pectic polysaccharides in pea epidermal cell walls

Polarized infrared absorption spectra of film specimens of theepidermal cell wall of the third internode of pea stems wererecorded before and after treatment with endopolygalacturonase(endo-PG) and endo-pectin lyase (endo-PL). The spectra showedthat the pectic polysaccharides solubilized with endo-PG wereessentially the same as those solubilized with endo-PL. Thedegree of esterification of the pectic polysaccharides was about20%, and their major sugar components were uronic acids (32.8%),arabinose (48.1%) and galactose (19.2%). The polarized infraredspectra showed that pectic polysaccharides have an orientedstructure in cell walls with their molecular chains orientedpreferentially parallel to the direction of cell elongation. ¹Present address: Research and Development, Kanzaki Paper Mfg.Co., Ltd., Amagasaki, Hyogo 660, Japan. ²Present address: Wakayama Research Laboratories, Kao Soap Co.,Ltd., Wakayama 640-91, Japan. (Received June 28, 1980; )     

Regulation by Abscisic Acid of In Vitro Flower Formation in Torenia Stem Segments

In Torenia stem segments cultured on a defined medium withoutphytohormones, in vitro flower formation was influenced by thephysiological states of the explants. Endogenous contents ofABA, but not those of IAA, were closely correlated with thephysiological states of the explants. Application of ABA (100ng/ml) to the culture medium stimulated flower formation inthe originally vegetative explants which otherwise had littleflower-forming capacity. Thus, endogenous ABA seems to be oneof the factors controlling the flower-forming capacity of Toreniastem segments. The highest rate of flower formation in the stemsegments was obtained when endogenous contents of ABA (whichresulted from both endogenously present and externally appliedABA) in the stem tissues was between 16 and 20 ng/g fresh weight. ¹ Present address: Bioscience Research Center, Mitsui PetrochemicalIndustries Ltd., Waki-cho, Kuga-gun, Yamaguchi 740, Japan. (Received November 22, 1984; Accepted March 1, 1985)     

PURIFICATION AND PROPERTIES OF SERINE HYDROXYMETHYLTRANSFERASE FROM NICOTIANA RUSTICA L.

Serine hydroxymethyltransferase was partially purified fromNicotiana rustica roots. The pH optimum for the enzyme is 4.0.A requirement for pyridoxal phosphate and a divalent cationwas shown, and the enzyme was inhibited by sulfhydryl reagentsand the reaction is folic acid-dependent. ¹Contribution from the Department of Biochemistry and publishedwith the approval of the Director of Research as Paper No. 2175in the Journal Series ²Present address; Spruance Research Laboratories, E. I. du Pontde Nemours and Company, Inc., Post Office Box 1477, Richmond,Virginia 23212, U.S.A.     

Some Characteristics of Membrane-Associated Protein Kinase in Lemna paucicostata

A protein kinase which phosphorylates histone was isolated fromthe endoplasmic reticulum-rich fractions of Lemna paucicostata.The enzyme could be solubilized by sonication, and its molecularweight was estimated as 220,000 by Sephacryl S-300 gel filtration.The optimum pH for enzyme activity was 9.0–9.5 and theactivity was stimulated by Co^2$, Mg^2$ and Mn^2$. Substrate proteinswhich might be phosphorylated by this protein kinase were alsodetected in microsomal fractions of Lemna plants. ¹ Present address: Advanced Research Laboratory, HITACHI LTD.,Kokubunji, Tokyo 185, Japan.     

Differential effect of Calcium and Magnesium on Mechanical Properties of Pea Stem Cell Walls

Replacement of calcium ion with magnesium ion in the cell wallof the pea epicotyl makes the wall more extensible. A possiblerole of this differential effect of Ca²⁺ and Mg^2– in regulatingcell elongation in pea epicotyl is discussed. The ratio of the content of calcium ion to that of magnesiumion (Ca²⁺/Mg²⁺) in the walls decreased markedly in the orderof the first > the second > the third internodes of thepea epicotyl. The capacity of the walls for cation exchangeincreased in the same order, whereas the calcium-magnesium ionexchange selectivity of the walls was virtually constant. Ourresults indicate that the changes in the Ca²⁺/Mg²⁺ ratio amongthe internodes is not attributable to the ion exchange propertiesof the walk per se, but is due to other physiological conditionswhich regulate the activities of free calcium and magnesiumions in the environment with which the walls are in equilibrium. ¹ Present address: Wakayama Research Laboratories, Kao SoapCo., Ltd., Wakayama 640-91, Japan ² Present address: Foold Development Laboratories, Meiji SeikaKaisha Ltd., Kawasaki 210, Japan (Received May 1, 1981; Accepted August 26, 1981)     

Sodium Stimulates Regeneration of Phosphoenolpyruvate in Mesophyll Chloroplasts of Amaranthus tricolor

Mesophyll chloroplasts were isolated from leaves of a Na⁺-requiringNAD-malic enzyme type, dicotyledonous C₄ plant, Amaranthus tricolorL. The chloroplasts converted pyruvate to phosphoenolpyruvateunder illumination, and the conversion was stimulated by Na⁺.This observation may explain the requirement for Na⁺ of someC₄ plants. ² Present address: Institute for Life Science Research, NihonNohyaku Co., Ltd., Kawachi-Nagano, Osaka, 586 Japan     

Sites of electron donation by alkylhydroquinones in the electron transport chain of spinach chloroplasts

Alkyl derivatives of p-hydroquinones were examined as electrondonors for the electron transport chain in spinach chloroplasts.Hydrophobic hydroquinones with long side chains donate relativelymore electrons to photosystem 1, while hydrophilic hydroquinoneand methylhydroquinone donate electrons specifically to photosystem2. ¹On leave; Research Laboratories, Fuji Photo Film Co., Ltd.,Asaka, Saitama. (Received March 2, 1973; )     

Sodium Requirement of Monocotyledonous C4 Plants for Growth and Nitrate Reductase Activity

The effects of Na application on growth and nitrate reductaseactivity of seven C₄ plant species, Zea mays, Echinochloa crus-galli,Panicum miliaceum, Panicum coloratum, Panicum dichotomiflorum,Panicum maximum and Chloris gayana were studied. Except forZ. mays and P. miliaceum, Na application enhanced growth significantly,and concurrent increases in nitrate reductase activities weredetected in Panicum coloratum, Panicum dichotomiflorum, Panicummaximum and Chloris gayana. ¹Present address: International Research Institute, Ciba GeigyJapan Ltd., Takarazuka, Hyogo 665, Japan. ²Present address: Photobiology Lab., Research Institute forFood Science, Kyoto Univ., Uji, Kyoto 611, Japan. (Received May 2, 1988; Accepted August 22, 1988)     

Ammonia as an important nitrogen source for the photosynthetic growth of Rhodospirillum rubrum

The growth of Rhodospirillum rubrum strain K was poor in a glutamatemalate(G-3-X) medium under light-anaerobic conditions. The supplementof ammonia to the G-3-X medium remarkably stimulated the growth.No such stimulation by ammonia was observed under dark-aerobicconditions. The other bacterium, strain M, also showed a similartendency for the ammonia requirement, though its growth in theG-3-X medium was much more abundant as compared to that of strainK. Casamino acid was found to contain stimulating factors for growth.Among the amino acids, arginine and/or histidine were effective.The other factor in casamino acid was found to be ammonia. The crude extracts of strains K and M contained glutamic dehydrogenaseas judged by the formation of glutamate from -ketoglutarateand ammonia. The highly reduced state of the cell componentsunder lightanaerobic conditions, appears to limit the formationof ammonia from glutamate. Ammonia was a preferable nitrogensource to glutamate under the conditions employed. ¹Present address: Institute for Agricultural Research, TohokuUniversity, Sendai. ²Present address: Sanitary Engineering Research Laboratory,Sanitary Engineering Department, Sumitomo Machinery Co. Ltd.,Hiratsuka.     

Reduced Permeability to K+ and Na+ Ions of K+ Channels in the Plasma Membrane of Tobacco Cells in Suspension after Adaptation to 50 mM NaCl

The whole-cell patch-clamp technique was used to study and comparethe characteristics of K⁺-and Na⁺-transport processes acrossthe plasma membrane in two types of protoplast isolated fromNaCl-adapted and -unadapted cells of tobacco (Nicotiana tabacumL. cv. Bright Yellow-2) in suspension culture. In both typesof protoplast, with 100 mM KCl in the bathing solution and inthe pipette solution, depolarization of the plasma membranefrom the holding potential of 0 mV to a positive potential resultedin a relatively large outward current which increased with increasingpositive potential, whereas hyperpolarization to negative potentialsup to –100 mV resulted in only a small inward current.The outward current activated by depolarization was predominantlycarried by K⁺ ions through K⁺ channels. Na⁺ ions also had afinite ability to pass through these K⁺ channels. The outwardK⁺ and Na⁺ currents of the NaCl-adapted cells were considerablysmaller than those of the NaCl-unadapted cells. These resultssuggest that adaptation to salinity results in reduced permeabilityof the plasma membrane to both K⁺ and Na⁺ ions. ¹Present address: Research Laboratory of Applied Biochemistry,Tanabe Seiyaku Co., Ltd., 16-89, Kashima 3-chome, Yodogawa-ku,Osaka, 532 Japan